Ligands specific for cannabinoid receptor subtype 2

ABSTRACT

A compound of Formula I: (I) has activity as a cannabinoid receptor antagonist. In Formula 1, R1 is unsubstituted or substituted aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aralkyl, or unsubstituted or substituted heteroaryl; R2 is unsubstituted or substituted alkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; and R3 is unsubstituted or substituted alkyl, unsubstituted or substituted aralkyl, or unsubstituted or substituted heteroaralkyl; with the proviso that at least one of Ri and R3 is other than unsubstituted aralkyl or R2 is other than unsubstituted aryl.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/984,461, filed Nov. 1, 2007, incorporated herein by reference in itsentirety.

GOVERNMENT RIGHTS

The United States Government has rights in this invention pursuant toContract No. NIH R01 DA015147 between the National Institutes of Healthand the University of Pittsburgh.

FIELD OF THE INVENTION

The present invention relates to compounds that bind with specificity tocannabinoid receptor subtype-2 (CB2).

BACKGROUND OF THE INVENTION

Cannabinoid receptor subtypes CB1 (brain) and CB2 (spleen) constitute acategory of the important GPCRs drug targets. CB receptors have beenimplicated in pain transduction and perception as well asneuroinflammation and autoimmune disorders. Finn et al., CurrentNeuropharmacology 2004, 2(1):75-89; and Walter et al., British Journalof Pharmacology 2004, 141(5): 775-85. In fact, natural marijuana-derivedcannabinoids have been used to relieve pain for millennia. Researchpublications have demonstrated the analgesic, anti-hyperalgesic andanti-inflammatory actions of selective CB 1 receptor agonists. Howlettet al., Neuropharmacology 2004, 47(Suppl. 1): 345-58. Activation of CB1receptors results, however, in sedation and undesirable psychotropiceffects. Efforts have been made to design CB2 ligands that selectivelyagonize CB2 receptors to treat chronic pain and neuronal disorderdiseases without the undesirable effects associated with activation ofCB1 receptors in the brain. Malan et al., Current Opinion inPharmacology 2003, 3(1): 62-67.

The CB2 receptor was initially cloned from macrophages and was found toexist predominantly in peripheral areas of the body enriched withB-lymphocytes (e.g., in spleen and lymph nodes). Munro et al., Nature(London) 1993, 365(64411): 61-65. The level of CB2 expression in varioustypes of inflammatory cells and immune competent cells is 10-100 timeshigher than CB1 receptor in these cell types. Galiegue et al., Eur JBiochem 1995, 232(1): 54-61; and Carlisle et al., InternationalImmunopharmacology 2002, 2(1): 69-82. Cannabinoids exhibitimmunosuppressive properties by interfering with humoral immunity,cell-mediated immunity, and cellular defenses against infectious agents.Berdyshev Chemistry and Physics of Lipids 2000, 108(1-2): 169-90.Overall, the emerging literature reveals the important roles of the CB2system and CB2 ligands on immune modulation.

Therapeutic potential of CB ligands has been shown in multiple reviews.Whiteside et al., Current Medicinal Chemistry 2007, 14(8): 917-36;Huffman J. W., Mini-Reviews in Medicinal Chemistry 2005, 5(7): 641-49;Pertwee R. G., Pharmacology & Therapeutics 2002, 95(2): 165-74; Hall etal., Lancet Oncology 2005, 6(1): 35-42; Howlett et al.,Neuropharmacology 2004, 47(Suppl. 1): 345-58; and Carter et al.,Physical Medicine and Rehabilitation Clinics of North America 2004,15(4): 943-54, ix. Although CB2 selective compounds have been limited inthe clinic, the cannabinoids could potentially be useful in treatingautoimmune and immunological disorders (e.g. multiple sclerosis). Theyhave also been studied as anti-inflammatory agents that alleviateinflammatory pain, Mbvundula et al., Inflammopharmacology 2004, 12(2):99-114; Malan et al., Current Opinion in Pharmacology 2003, 3(1): 62-67;and Cravatt et al., Journal of Neurobiology 2004, 61(1): 149-60, asanti-cancer agents that inhibit the growth of tumors of immune origin aswell as glioma tumors and non-melanoma skin cancers, McKallip et al.,Blood 2002, 100(2): 627-34, and as agents to induce apoptosis in immunesystem cancer. Bifulco et al., Recenti Progressi In Medicina 2003,94(5):194-98.

It is apparent that the CB2 receptor has a complex involvement involvedwith the immune system, with tumor cells, and with inflammation. Thediscovery of CB2 receptors in the CNS contrasts with previous efforts todetect non-CB1 receptors in brain tissue. Van Sickle et al., Science2005, 310(5746): 329-32. The purpose of these receptors beyond immunecell regulation remains controversial. Still, these studies did open thepossibility of non-psychotropic therapeutic interventions, usingenhanced endocannabinoid levels localized in the brain, and they offertherapeutic promise for treating central nervous system disorders.Whiteside et al., Current Medicinal Chemistry 2007, 14(8): 917-36.

In summary, available evidence suggests that cannabinoids could bevaluable, particularly as adjuvants for symptom control in a range ofconditions for which standard drugs are not fully satisfactory. AshtonAddict. Biol. 1999, 4(2): 111-26. Work has been limited, however, inrelation to the design of CB2 ligands that do not confer psychotropicside effects. The slow pace of development in this regard has been duelargely to a lack of information about the three-dimensional structuresof the CB receptors and ligand binding sites.

Accordingly, need exists for CB2 chemical probes that can be used todistinguish the CB1 and CB2 pharmacophore features for structure-baseddesign, and to identify novel CB2 selective ligands with the potentialfor development into therapeutic agents that have no psychotropic sideeffects.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, compounds areprovided that possess the appropriate chemical scaffolds for selectiveCB2 biological selectivity. In one embodiment, a compound Formula I, apharmaceutically acceptable salt of the compound of Formula I, astereoisomer of the compound of Formula I, or a pharmaceuticallyacceptable salt of the stereoisomer of the compound of Formula I, areprovided:

wherein;

-   -   R₁ is unsubstituted or substituted alkyl, unsubstituted or        substituted aryl, unsubstituted or substituted cycloalkyl,        unsubstituted or substituted cycloalkylalkyl, unsubstituted or        substituted heterocyclyl, unsubstituted or substituted        heterocyclylalkyl, unsubstituted or substituted aralkyl,        unsubstituted or substituted heteroaryl, or unsubstituted or        substituted heteroaralkyl;    -   R₂ is unsubstituted or substituted alkyl, unsubstituted or        substituted aryl, or unsubstituted or substituted heteroaryl;        and    -   R₃ is unsubstituted or substituted alkyl, unsubstituted or        substituted aryl, unsubstituted or substituted cycloalkyl,        unsubstituted or substituted cycloalkylalkyl, unsubstituted or        substituted heterocyclyl, unsubstituted or substituted        heterocyclylalkyl, unsubstituted or substituted aralkyl,        unsubstituted or substituted heteroaryl, or unsubstituted or        substituted heteroaralkyl;    -   with the proviso that at least one of R₁ and R₃ is other than        unsubstituted aralkyl, or R₂ is other than unsubstituted aryl.

In some embodiments of the compound of Formula I,

-   -   R₁ is a substituted or unsubstituted group selected from the        group consisting of phenyl or naphthyl; cyclohexyl, cycloheptyl,        cyclooctyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl,        bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.2.2]nonyl,        bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decanyl, adamantyl,        noradamantyl, bornyl, or norbornyl; pyrrolidinyl,        imidazolidinyl, pyrazolidinyl, pyrrolyl, pyrrolinyl, imidazolyl,        imidazolinyl, pyrazolyl, pyrazolinyl, piperidyl, piperazinyl,        morpholinyl, thiomorpholinyl, pyranyl, quinuclidyl, indolyl,        indolinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl,        quinolizinyl, quinoxalinyl, or quinazolinyl; —(C1-C8        alkyl)phenyl; and pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,        or benzothiophenyl;    -   R₂ is a substituted or unsubstituted group selected from the        group consisting of C1-C8 alkyl; phenyl, naphthyl, and pyridyl;        and/or    -   R₃ is a substituted or unsubstituted group selected from the        group consisting of C1-C8 alkyl, —(C1-C8 alkyl)phenyl, and        —(C1-C8 alkyl)pyridyl.

In certain embodiments, R₁ of Formula I is substituted or unsubstitutedgroup selected from the group consisting of phenyl,bicyclo[2.2.1]heptyl, benzyl, and morpholinyl; and/or R₂ is asubstituted or unsubstituted group selected from the group consisting ofpropyl, butyl, and phenyl; and/or R₃ is a substituted or unsubstitutedgroup selected from the group consisting of propyl, butyl, and benzyl.In other embodiments, the compound of Formula I has a group that issubstituted is substituted with one or more of H, F, Cl, Br, SO₂, NO₂,OH, NH₂, or substituted or unsubstituted C₁-C₈ alkyl.

Pursuant to another aspect of the invention, the compound of Formula Iis selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, a stereoisomer thereof,or a pharmaceutically acceptable salt of the stereoisomer thereof.

The present invention also comprehends a pharmaceutical formulationsthat comprises (A) a pharmaceutically acceptable carrier and (B) acompound of Formula I, a pharmaceutically acceptable salt of suchcompound, a stereoisomer of the compound, a pharmaceutically acceptablesalt of such stereoisomer, a compound of Formula II, a pharmaceuticallyacceptable salt of the compound of Formula II, or a mixture of any twoor more thereof. Formula II is shown below:

In another aspect, a method is provided that comprises inhibiting acannabinoid receptor comprising administering a pharmaceuticalformulation comprising a compound of Formula I, a pharmaceuticallyacceptable salt of the compound of Formula I, a stereoisomer of thecompound of Formula I, a pharmaceutically acceptable salt of thestereoisomer of the compound of Formula I, Formula II, apharmaceutically acceptable salt of the compound of Formula II, or amixture of any two or more thereof, and a pharmaceutically acceptablecarrier, to a subject. In some such embodiments, the cannabinoidreceptor is cannabinoid receptor subtype-2. In other embodiments, thesubject is in need of an anti-inflammatory agent.

In yet another aspect, methods are provided to determine the 3Dstructure and conformation of CB2 ligands. For example, in someembodiments, a method is provided comprising, using a compound ofFormula I, a pharmaceutically acceptable salt of the compound of FormulaI, a stereoisomer of the compound of Formula I, a pharmaceuticallyacceptable salt of the stereoisomer of the compound of Formula I,Formula II, a pharmaceutically acceptable salt of the compound ofFormula II, or a mixture of any two or more thereof as an activefluorescence dye control for a fluorometric binding assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents two graphs of radiometric binding assay studies, showingthat the compound of Formula II has high CB2 binding affinity, CB2 (31.7nM) (A), but weak CB1 binding affinity, CB1 (4185 nM) (B). FIG. 1A isCB2 binding ³H—CP 55940 (mouse spleen membrane) and FIG. 1B is CB1binding ³H—CP 55940 (rat forebrain membrane).

FIG. 2. Confluentially growing CB2 receptor transfected human CHO-K1cells were stimulated with forskolin (20 μM) and analyzed for cAMPexpression after 30 min. The CB2 agonist WIN55, 212-2 was incubated atincreasing concentrations 30 min prior to forskolin stimulation alone orin presence of 1 μM of the antagonists AM630 and the compound of FormulaII. The compound of Formula II, like AM630, inhibits the effect ofWIN55,212-2.

FIG. 3. Confluentially growing CB2 receptor transfected human CHO-K1cells were stimulated with forskolin (20 μM) and analyzed for cAMPexpression after 30 minutes. CB2 ligands were incubated at increasingconcentrations 30 minutes prior to forskolin stimulation. AM630 andSR144528 are inverse agonist. The compound of Formula II is a weakagonist at high concentration (>7 mM) but also a antagonist whenincubated with WIN55, 212-2, see FIG. 2.

DETAILED DESCRIPTION

Definitions

For this disclosure and unless otherwise specified, “a” or “an” means“one or more.”

As indicated above, “CB” is an abbreviation for “cannabinoid receptor.”Similarly, “CB1” stands for “cannabinoid receptor subtype-1,” and “CB2”for “cannabinoid receptor subtype-2.”

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Accordingly, isotopically labeled compounds are within thescope of this invention.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup will be substituted with one or more substituents, unlessotherwise specified. In some embodiments, a substituted group issubstituted with 1, 2, 3, 4, 5, or 6 substituents. Examples ofsubstituent groups include: halogens (i.e., F, Cl, Br, and I);hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy,heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines;aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls;sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones;azides; amides; ureas; amidines; guanidines; enamines; imides;isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitrogroups; nitriles (i.e., CN); and the like.

Substituted ring groups such as substituted cycloalkyl, aryl,heterocyclyl and heteroaryl groups also include rings and fused ringsystems in which a bond to a hydrogen atom is replaced with a bond to acarbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl andheteroaryl groups may also be substituted with substituted orunsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

Alkyl groups include straight chain and branched alkyl groups havingfrom 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkylgroups further include cycloalkyl groups as defined below. Examples ofstraight chain alkyl groups include those with from 1 to 8 carbon atomssuch as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,and n-octyl groups. Examples of branched alkyl groups include, but arenot limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl,isopentyl, and 2,2-dimethylpropyl groups. Representative substitutedalkyl groups may be substituted one or more times with substituents suchas those listed above.

Cycloalkyl groups are cyclic alkyl groups such as, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8ring members, whereas in other embodiments the number of ring carbonatoms range from 3 to 5, 3 to 6, or 3 to 7. Cycloalkyl groups furtherinclude mono-, bicyclic and polycyclic ring systems, such as, forexample bridged cycloalkyl groups as described below, and fused rings,such as, but not limited to, decalinyl, and the like. In someembodiments, polycyclic cycloalkyl groups have three rings. Substitutedcycloalkyl groups may be substituted one or more times with,non-hydrogen and non-carbon groups as defined above. However,substituted cycloalkyl groups also include rings that are substitutedwith straight or branched chain alkyl groups as defined above.Representative substituted cycloalkyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, 2,2-, 2,3-,2,4-2,5- or 2,6-disubstituted cyclohexyl groups, which may besubstituted with substituents such as those listed above.

Bridged cycloalkyl groups are cycloalkyl groups in which two or morehydrogen atoms are replaced by an alkylene bridge, wherein the bridgecan contain 2 to 6 carbon atoms if two hydrogen atoms are located on thesame carbon atom, or 1 to 5 carbon atoms, if the two hydrogen atoms arelocated on adjacent carbon atoms, or 2 to 4 carbon atoms if the twohydrogen atoms are located on carbon atoms separated by 1 or 2 carbonatoms. Bridged cycloalkyl groups can be bicyclic, such as, for examplebicyclo[2.1.1]hexane, or tricyclic, such as, for example, adamantyl.Representative bridged cycloalkyl groups include bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl,bicyclo[3.2.2]nonyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decanyl,adamantyl, noradamantyl, bornyl, or norbornyl groups. Substitutedbridged cycloalkyl groups may be substituted one or more times withnon-hydrogen and non-carbon groups as defined above. Representativesubstituted bridged cycloalkyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di- ortri-substituted adamantyl groups, which may be substituted withsubstituents such as those listed above.

Cycloalkylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to acycloalkyl group as defined above. In some embodiments, cycloalkylalkylgroups have from 4 to 20 carbon atoms, 4 to 16 carbon atoms, andtypically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups maybe substituted at the alkyl, the cycloalkyl or both the alkyl andcycloalkyl portions of the group. Representative substitutedcycloalkylalkyl groups may be mono-substituted or substituted more thanonce, such as, but not limited to, mono-, di- or tri-substituted withsubstituents such as those listed above.

Alkenyl groups include straight and branched chain and cycloalkyl groupsas defined above, except that at least one double bond exists betweentwo carbon atoms. Thus, alkenyl groups have from 2 to about 20 carbonatoms, and typically from 2 to 12 carbons or, in some embodiments, from2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkenylgroups include cycloalkenyl groups having from 4 to 20 carbon atoms, 5to 20 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbonatoms. Examples include, but are not limited to vinyl, allyl,—CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂, —C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂,cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl,and hexadienyl, among others. Representative substituted alkenyl groupsmay be mono-substituted or substituted more than once, such as, but notlimited to, mono-, di- or tri-substituted with substituents such asthose listed above.

Cycloalkenylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of the alkyl group is replaced with a bond to acycloalkenyl group as defined above. Substituted cycloalkylalkenylgroups may be substituted at the alkyl, the cycloalkenyl or both thealkyl and cycloalkenyl portions of the group. Representative substitutedcycloalkenylalkyl groups may be substituted one or more times withsubstituents such as those listed above.

Alkynyl groups include straight and branched chain alkyl groups, exceptthat at least one triple bond exists between two carbon atoms. Thus,alkynyl groups have from 2 to about 20 carbon atoms, and typically from2 to 12 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4carbon atoms. Examples include, but are not limited to —C≡CH, —C═C(CH₃),—C═C(CH₂CH₃), —CH₂C═CH, —CH₂C═C(CH₃), and —CH₂C═C(CH₂CH₃), among others.Representative substituted alkynyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di- ortri-substituted with substituents such as those listed above.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups include monocyclic, bicyclic and polycyclicring systems. Thus, aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl,indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments,aryl groups contain 6-14 carbons, and in others from 6 to 12 or even6-10 carbon atoms in the ring portions of the groups. Although thephrase “aryl groups” includes groups containing fused rings, such asfused aromatic-aliphatic ring systems (e.g., indanyl,tetrahydronaphthyl, and the like), it does not include aryl groups thathave other groups, such as alkyl or halo groups, bonded to one of thering members. Rather, groups such as tolyl are referred to assubstituted aryl groups. Representative substituted aryl groups may bemono-substituted or substituted more than once. For example,monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-,5-, or 6-substituted phenyl or naphthyl groups, which may be substitutedwith substituents such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined above. In some embodiments, aralkyl groups contain 7 to 20carbon atoms, 7 to 14 carbon atoms or 7 to 10 carbon atoms. Substitutedaralkyl groups may be substituted at the alkyl, the aryl or both thealkyl and aryl portions of the group. Representative aralkyl groupsinclude but are not limited to benzyl and phenethyl groups and fused(cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Representativesubstituted aralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, heterocyclyl groups include 3 to 20 ringmembers, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3to 15 ring members. Heterocyclyl groups encompass unsaturated, partiallysaturated and saturated ring systems, such as, for example, imidazolyl,imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group”includes fused ring species including those comprising fused aromaticand non-aromatic groups, such as, for example, benzotriazolyl,2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase alsoincludes bridged polycyclic ring systems containing a heteroatom suchas, but not limited to, quinuclidyl. However, the phrase does notinclude heterocyclyl groups that have other groups, such as alkyl, oxoor halo groups, bonded to one of the ring members. Rather, these arereferred to as “substituted heterocyclyl groups.” Heterocyclyl groupsinclude, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl,tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl,imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl,thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl,thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane,dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl,pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl,dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl,isoindolyl, azaindolyl(pyrrolopyridyl), indazolyl, indolizinyl,benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl,benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl,imidazopyridyl(azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl,purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl,quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl,naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl,dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl,tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl,tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl,tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl,tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups may be mono-substitutedor substituted more than once, such as, but not limited to, pyridyl ormorpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, ordisubstituted with various substituents such as those listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S. Heteroaryl groups include, but are not limited to,groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl,azaindolyl(pyrrolopyridyl), indazolyl, benzimidazolyl,imidazopyridyl(azabenzimidazolyl), pyrazolopyridyl, triazolopyridyl,benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,imidazopyridyl, isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl,adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,quinoxalinyl, and quinazolinyl groups. Although the phrase “heteroarylgroups” includes fused ring compounds such as indolyl and 2,3-dihydroindolyl, the phrase does not include heteroaryl groups that have othergroups bonded to one of the ring members, such as alkyl groups. Rather,heteroaryl groups with such substitution are referred to as “substitutedheteroaryl groups.” Representative substituted heteroaryl groups may besubstituted one or more times with various substituents such as thoselisted above.

Heterocyclylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheterocyclyl group as defined above. Substituted heterocyclylalkylgroups may be substituted at the alkyl, the heterocyclyl or both thealkyl and heterocyclyl portions of the group. Representativeheterocyclyl alkyl groups include, but are not limited to,4-ethyl-morpholinyl, 4-propylmorpholinyl, furan-2-yl methyl, furan-3-ylmethyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-ylpropyl. Representative substituted heterocyclylalkyl groups may besubstituted one or more times with substituents such as those listedabove.

Heteroaralkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above. Substituted heteroaralkyl groups maybe substituted at the alkyl, the heteroaryl or both the alkyl andheteroaryl portions of the group. Representative substitutedheteroaralkyl groups may be substituted one or more times withsubstituents such as those listed above.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the inventionare designated by use of the suffix, “ene.” For example, divalent alkylgroups are alkylene groups, divalent aryl groups are arylene groups,divalent heteroaryl groups are divalent heteroarylene groups, and soforth. Substituted groups having a single point of attachment to thecompound of the invention are not referred to using the “ene”designation. Thus, chloroethyl is not referred to here as“chloroethylene.”

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of a substituted orunsubstituted alkyl group as defined above. Examples of linear alkoxygroups include but are not limited to methoxy, ethoxy, propoxy, butoxy,pentoxy, hexoxy, and the like. Examples of branched alkoxy groupsinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groupsinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. Representative substitutedalkoxy groups may be substituted one or more times with substituentssuch as those listed above.

The terms “aryloxy” and “arylalkoxy” refer to, respectively, asubstituted or unsubstituted aryl group bonded to an oxygen atom and asubstituted or unsubstituted aralkyl group bonded to the oxygen atom atthe alkyl. Examples include but are not limited to phenoxy, naphthyloxy,and benzyloxy. Representative substituted aryloxy and arylalkoxy groupsmay be substituted one or more times with substituents such as thoselisted above.

The term “amine” (or “amino”) as used herein refers to —NHR⁴ and —NR⁵R⁶groups, wherein R⁴, R⁵ and R⁶ are independently hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Insome embodiments, the amine is NH₂, methylamino, dimethylamino,ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, orbenzylamino.

Those of skill in the art will appreciate that compounds of theinvention may exhibit the phenomena of tautomerism, conformationalisomerism, geometric isomerism and/or optical isomerism. As the formuladrawings within the specification and claims can represent only one ofthe possible tautomeric, conformational isomeric, optical isomeric orgeometric isomeric forms, it should be understood that the inventionencompasses any tautomeric, conformational isomeric, optical isomericand/or geometric isomeric forms of the compounds having one or more ofthe utilities described herein, as well as mixtures of these variousdifferent forms.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The concentrations of the isomeric formswill depend on the environment the compound is found in and may bedifferent depending upon, for example, whether the compound is a solidor is in an organic or aqueous solution. For example, in aqueoussolution, pyrazoles may exhibit the following isomeric forms, which arereferred to as tautomers of each other:

As readily understood by one skilled in the art, a wide variety offunctional groups and other structures may exhibit tautomerism, and alltautomers of compounds as described herein are within the scope of thepresent invention.

Stereoisomers of compounds, also known as “optical isomers,” include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present invention include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these are all withinthe scope of the invention.

“A pharmaceutically acceptable carrier” is a phrase that denotes acarrier such as but not limited to a diluent, an excipient, a wettingagent, a buffering agent, a suspending agent, a lubricating agent, anadjuvant, a vehicle, a delivery system, an emulsifier, a disintegrant,an absorbent, a preservative, a surfactant, a colorant, a flavorant, asweetener, or a mixture of any two or more thereof. Pharmaceuticallyacceptable excipients and carriers are generally known and, hence, areincluded in the instant invention. Such materials are described, forexample, in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21^(st)ed., The University of Philadelphia (2005).

The instant invention also provides for pharmaceutical compositions andmedicaments which may be prepared by mixing one or more compounds of theinvention, prodrugs thereof, pharmaceutically acceptable salts thereof,stereoisomers thereof, tautomers thereof, or solvates thereof, withpharmaceutically acceptable carriers, excipients, binders, diluents orthe like to prevent and treat disorders associated with cannabinoidreceptors. The compounds and compositions of the invention may be usedto prepare formulations and medicaments that prevent or treat a varietyof disorders associated with cannabinoid receptors, and describedherein. For example, disorders and diseases such as obesity, smokingaddiction, cardimetabolic risk factors, and other disorder and diseasesassociated with the central nervous system.

Illustrative of the forms suitable for such compositions are granules,powders, tablets, capsules, syrup, suppositories, injections, emulsions,elixirs, suspensions or solutions. A composition of the invention can beformulated for various routes of administration, for example, by oral,parenteral, topical, rectal, nasal, or vaginal administration, or viaimplanted reservoir. Parenteral or systemic administration includes butis not limited to subcutaneous, intravenous, intraperitoneally,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injections. The followingdosage forms are given by way of example and should not be construed aslimiting the instant invention.

As noted, the invention comprehends pharmaceutically acceptable salts ofthe compounds described here. A compound of the invention has a numberof basic nitrogen groups; hence, pharmaceutically acceptable salts canbe formed with inorganic acids (such as hydrochloric acid, hydroboricacid, nitric acid, sulfuric acid, and phosphoric acid), organic acids(e.g., formic acid, acetic acid, trifluoroacetic acid, fumaric acid,oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid,succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid,and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acidand glutamic acid). The compounds of the present invention may haveacidic substituent groups, and in such cases, it can form salts withmetals, such as alkali and earth alkali metals (e.g., Na⁺, Li⁺, K⁺,Ca²⁺, Mg²⁺, Zn²⁺), organic amines (e.g., ammonia, trimethylamine,triethylamine, pyridine, picoline, ethanolamine, diethanolamine,triethanolamine) or basic amino acids (e.g. arginine, lysine andornithine).

Certain compounds within the scope of the invention are derivativesreferred to as prodrugs. The expression “prodrug” denotes a derivativeof a known direct acting drug, e.g. esters and amides, which derivativehas enhanced delivery characteristics and therapeutic value as comparedto the drug, and is transformed into the active drug by an enzymatic orchemical process; see Notari, R. E., Methods in Enzymology 112: 309-23(1985); Bodor, N., Drugs of the Future 6: 165-82 (1981); and Bundgaard,H., “Design of Prodrugs: Bioreversible-Derivatives for VariousFunctional Groups and Chemical Entities,” in DESIGN OF PRODRUGS (H.Bundgaard, ed.), Elsevier (1985), Goodman and Gilmans, THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 8th ed., McGraw-Hill (1992).

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the instant invention, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives are sucrose, lactose,cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates,chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,collagens, casein, albumin, synthetic or semi-synthetic polymers orglycerides. Optionally, oral dosage forms can contain other ingredientsto aid in administration, such as an inactive diluent, or lubricantssuch as magnesium stearate, or preservatives such as paraben or sorbicacid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Tablets and pills may be furthertreated with suitable coating materials known in the art.

Compounds

In one aspect, a compound of Formula I is provided, having the followingstructural formula:

In the compound of Formula I, R₁ is unsubstituted or substituted alkyl,unsubstituted or substituted aryl, unsubstituted or substitutedcycloalkyl, unsubstituted or substituted cycloalkylalkyl, unsubstitutedor substituted heterocyclyl, unsubstituted or substitutedheterocyclylalkyl, unsubstituted or substituted aralkyl, unsubstitutedor substituted heteroaryl, or unsubstituted or substitutedheteroaralkyl. In the compound of Formula I, R₂ is unsubstituted orsubstituted alkyl, unsubstituted or substituted aryl, or unsubstitutedor substituted heteroaryl. In the compound of Formula I, R₃ isunsubstituted or substituted alkyl, unsubstituted or substituted aryl,unsubstituted or substituted cycloalkyl, unsubstituted or substitutedcycloalkylalkyl, unsubstituted or substituted heterocyclyl,unsubstituted or substituted heterocyclylalkyl, unsubstituted orsubstituted aralkyl, unsubstituted or substituted heteroaryl, orunsubstituted or substituted heteroaralkyl. The compound of Formula I isalso subject to the proviso, that at least one of R₁ and R₃ is otherthan unsubstituted aralkyl, or R₂ is other than unsubstituted aryl.

In some embodiments, R₁ is a substituted or unsubstituted group such asphenyl, naphthyl, cyclohexyl, cycloheptyl, cyclooctyl,bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl,bicyclo[2.2.2]octyl, bicyclo[3.2.2]nonyl, bicyclo[3.3.1]nonyl,bicyclo[3.3.2]decanyl, adamantyl, noradamantyl, bornyl, norbornyl,pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolyl, pyrrolinyl,imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, piperidyl,piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, quinuclidyl,indolyl, indolinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl,quinolizinyl, quinoxalinyl, quinazolinyl, —(C1-C8 alkyl)phenyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, or benzothiophenyl.

In other embodiments, R₂ is a substituted or unsubstituted group such asC₁-C₈ alkyl, phenyl, naphthyl, or pyridyl.

In yet other embodiments, R₃ is a substituted or unsubstituted groupsuch as C₁-C₈ alkyl, —(C₁-C₈ alkyl)phenyl, or —(C₁-C₈ alkyl)pyridyl.

A non-limiting illustration of the compounds encompassed by Formula I,and the associated isomeric/tautomeric forms, includes:

where the variable positions are defined in Table 1.

TABLE 1 No. R₁ R₂ R₃ Note 1

Cl position is variable CH₃ position is variable 2

Cl position is variable CH₃ position is variable 3

Chain length is variable Cl position is variable 4

Chain length is variable Cl position is variable 5

OH position is variable 6

OH position is variable 7

OH position is variable 8

Chain length is variable Cl position is variable 9

Chain length is variable Cl position is variable 10

Chain length is variable Cl position is variable 11

Chain length is variable Cl position is variable 12

Chain length is variable Cl position is variable 13

Chain length is variable Cl position is variable 14

Chain length is variable Cl position is variable 15

Chain length is variable Cl position is variable 16

Chain length is variable Cl position is variable

In some embodiments, the compound of Formula I is represented by FormulaIII, or a pharmaceutically acceptable salt of the compound of FormulaIII:

wherein R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉,R₂₀, and R₂₁ are individually H, F, Cl, Br, SO₂, NO₂, OH, NH₂, orsubstituted or unsubstituted C₁-C₈ alkyl. The compound of Formula III isalso subject to the proviso of the compound of Formula I, such that atleast one of R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈,R₁₉, R₂₀, and R₂₁ is other than H.

Compounds of the invention may be readily synthesized by techniques wellknown to those of skill in the art.

In another aspect, pharmaceutical compositions are provided. Forexample, pharmaceutical compositions of the compound of Formula I, apharmaceutically acceptable salt of the compound of Formula I, astereoisomer of the compound of Formula I, a pharmaceutically acceptablesalt of the stereoisomer of the compound of Formula I, a compound ofFormula II, a pharmaceutically acceptable salt of the compound ofFormula II, or a mixture of any two or more thereof, and apharmaceutically acceptable carrier are provided. The compound ofFormula II has the following structure:

In yet another aspect, methods inhibiting a cannabinoid receptor areprovided. Such methods may include administering the pharmaceuticalcompositions, provided herein, to a subject. Such subject may be in needof a cannabinoid receptor antagonist.

The compound of Formula II:

is a chemical probe with a high biological activity that selectivelybinds or interacts strongly with CB2. The compound of Formula II, hasbeen shown to bind specifically to CB2. Such reactivity was shown usingthe knowledge-based cannabinoid (CB) ligand design approach, i.e.,pharmacophore generation and in-silico database virtual screening aswell as bio-validations. The compound of Formula II demonstratesnanomolar (nM) binding bioaffinity to the cannabinoid receptor subtypeCB2.

In addition to binding CB, the compound of Formula II has been shown tobe an active fluorescence dye with maximum fluorescence intensity at 485nm. The fluorescence spectral measurements were done for the compound offormula II at 5.0 μM of a solution containing 25 mM Tris-HCl, 2.5 mMMgCl2, 1 mM EDTA. The sample was placed in a 3 ml fluorescence cuvette(excitation pathlength=10 mm) and spectrum recorded at 22° C. on a CaryEclipse Fluorescence Spectrophotometer (Varian Inc.). The maximumfluorescence intensity was observed at 485 nm when the excitationwavelength was set at 468 nm and emission wavelength was started from478 nm. The compounds of Formulas I and II fluoresce and may thereforebe used as fluorescence image probes in binding assays for CB receptors.As such, the compounds of Formula I and/or Formula II may be reactedwith a CB subtype 1 or 2 receptor, or a sample thought to contain a CBsubtype 1 or 2 receptor. After, sample processing, the sample may thenbe assayed for the presence of the compound of Formula I and/or FormulaII.

The compound of Formula II has strong CB2 specific binding activity andmay also be used as an effective anti-inflammatory agent. It is knownthat CB2 receptor mediates signal transduction in the immune system, andCB2 ligands have the potential to be developed as drugs to treat a widerange of immune system disorders as well as chronic neuropains. SeeCorrea F, Mestre L, et. al. The role of cannabinoid system on immunemodulation: Therapeutic implications on CNS inflammation. Mini-Reviewsin Medicinal Chemistry 2005, 5(7):671-675; Whiteside G T, et. al. Therole of the cannabinoid CB2 receptor in pain transmission andtherapeutic potential of small molecule CB2 receptor agonists. CurrentMedicinal Chemistry 2007, 14(8):917-936; and Howlett A C, et. al.Cannabinoid physiology and pharmacology: 30 years of progress.Neuropharmacology 2004, 47(Suppl. 1):345-358.

One skilled in the art will readily realize that all ranges discussedcan and do necessarily also describe all subranges therein for allpurposes and that all such subranges also form part and parcel of thisinvention. Any listed range can be easily recognized as sufficientlydescribing and enabling the same range being broken down into at leastequal halves, thirds, quarters, fifths, tenths, etc. As a non-limitingexample, each range discussed herein can be readily broken down into alower third, middle third and upper third, etc.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES

In-silico Virtual Screening. Virtual screening studies were carried outon a Dell Cluster System consisting of 30 dual cores/dual nodes Xenonprocessors. 3D-QSAR studies of bioactive antagonists of the CB receptorswere conducted, from which pharmacophore queries were developed by useof SPL scripts with DISCO and GALAHAD (Tripos programs) for 3D databasesearch. See Chen et al. 3D-QSAR Studies of Arylpyrazole Antagonists ofCannabinoid Receptor Subtypes CB1 and CB2. A Combined NMR and CoMFAApproach, Journal of Medicinal Chemistry 2006, 49(2):625-636. Thetraining database, consisting of 20 known active CB2 ligands and 980random compounds, was established to examine and/or refine the generatedpharmacophore hypotheses and to ensure the liability of the generatedpharmacophore models. Subsequently, 3D pharmacophore database searcheswere carried out based on the pharmacophore queries generated above. Thehit selections were done both manually and computationally in comparingwith the actual pharmacophore models by the established scoring functionand hit ranking algorithm. Such screening methods and other methods usedherein are more fully described in Chen et al., J. Chem. Inf. Model2007, 47: 1626-37. The compound of Formula II was one of the identifiedvirtually CB2-active compounds, and was then further biologicallyvalidated using in-vitro radiometric binding assay below.

In-vitro Bio-validation Experiment. To biologically validate thescreened hit compounds above, in-vitro [³H]-radioactive CB ligandbinding assays were carried out to determine the CB receptor bindingaffinity (Ki) of the screened ligands by displacing [³H]-CP-55,940. Thecompound of Formula II was obtained from National Cancer Institute(NCI). For each assay, a positive control with a known ligand(SR-141716A for CB1 and SR-144528 for CB2) was run to ensure thereliability of the assay.

For CB1 receptor binding studies, rat forebrain membranes were preparedfollowing the reported procedures. See Goutopoulos et al., Bioorganic &Medicinal Chemistry 2001, 9(7): 1673-84; and Mussinu, et. al.,Bioorganic & Medicinal Chemistry 2003, 11(2):251-263.

For CB2 receptor binding studies, membranes were prepared from frozenmouse spleen. See Goutopoulos et al., Bioorganic & Medicinal Chemistry2001, 9(7): 1673-84; Lin et al., J Med Chem 1998, 41(27): 5353-61; andChin et al., J. Pharmacol Exp Ther 1999, 291(2):837-844.

The experiments were conducted based on reported procedures. See Lan etal., AAPS PharmSci [online computer file] 1999, 1(3):article 4; Abadjiet al., J. Med. Chem. 1994, 37(120:1889-1893; and Abadji et al., J.Neurochem. 1999, 72(5):2032-38. Thus, the competition binding assay wasperformed using [3H]-CP-55,940 (1 nM) as a radioligand for the CB1 orCB2 cannabinoid receptor (rate brain or mouse spleen membranes), at 30°C. in 96-well microtiter plates, 50 μg of membrane each wellre-suspended in 200 μl (final volume) binding buffer (50 mM Tris-HCl,2.5 mM EGTA, 3 mM MgCl₂, 0.1% bovine serum albumin, pH 7.4). The testedcompounds were presented at varying concentrations, and the nonspecificbinding was determined in the presence of 10 μM CP-55,940 or HU-210.After 1 hr the incubation was terminated by rapid vacuum filtrationthrough 96-well GF/B filter plates (PerkinElmer) on a PerkinElmer cellharvester and washed 5 times with one well-volume of ice-cold washingbuffer (same as binding buffer except 0.5% bovine serum albumin). Thefilters were dried and the radioactivity on the filters was measured ina PerkinElmer TopCount Microplate Scintillation Counter using 30 μl ofMicroScint-20 PerkinElmer. Assays were performed in duplicate ortriplicate wells of the 96-well microtiter plates. The K_(i) values werecalculated by using GraphPad Prism 5.0 (GraphPad Software, Inc.).

Cell-based Functional Bioassay Studies. The nanomolar-binding affinityof the compound of Formula II have been illustrated using the³H-radiometric binding assay shown in FIG. 1. Subsequently, furtherreceptor biological studies were performed: i) to examine/assess theG-protein effects of the compound of Formula II upon CB2 receptorbinding and ii) to test the compound of Formula II for anti-inflammatoryeffects. These studies confirmed that the compound of Formula II appearsto be an antagonist like the known CB2 antagonist AM630 (but weaker),inhibiting the effect of the CB2 agonist WIN55212-2 in theforskolin-stimulated cAMP assay experiments as shown by FIG. 2.

Interestingly, however, the compound of Formula II is not an inverseagonist like the other known CB2 antagonists (e.g., AM630 and SR144528).The further cAMP measurement (FIG. 3) shows that the compound of FormulaII behaves as a real antagonist by blocking cAMP effects but becomes anagonist at higher (˜10 μM) concentrations.

The compound of Formula II acts as an antagonist by inhibiting cAMP butacts as a weak agonist at higher μM concentrations. Unlike the known CB2inverse agonists, our compound does not have an impact on Gi-proteins atconcentrations up to low μM and does not affect Go signaling. It shouldbe pointed out that such concentration-dependent dual antagonist-agonisteffects have also been reported for other receptors, e.g., neurotensinreceptor, acetylcholine receptor, 5-HT1 receptor, and dopamine receptor.Cusack et al., Molecular Pharmacology 1993, 44(5):1036-1040; Schlickeret al., Pharmacol Toxicol. 1988, 63(4):281-285; and Mulder et al., Eur.J. Pharmacol. 1985, 107(3):291-297. According to R. Seifert's book “GPCRas Drug Targets,” most GPCR antagonists on the market are inverseagonists but not true antagonists like our compound the compound ofFormula II. Thus, the compound of Formula II is a unique CB2 chemicalprobe and offers a new avenue to explore the CB2 ligand bindingbiological mechanism. Seifert et al., in METHODS PRINC. MED. CHEM., vol.24 (Wiley-VCH, 2005).

While several, non-limiting examples have been illustrated anddescribed, it should be understood that changes and modifications can bemade therein in accordance with ordinary skill in the art withoutdeparting from the invention in its broader aspects as defined in thefollowing claims.

What is claimed is:
 1. A compound of Formula I, a pharmaceuticallyacceptable of the compound of Formula I, a stereoisomer of the compoundof Formula I, a pharmaceutically acceptable salt of the stereoisomer ofthe compound of Formula I:

wherein; R₁ is unsubstituted or substituted alkyl, unsubstituted orsubstituted aryl, unsubstituted or substituted cycloalkyl, unsubstitutedor substituted cycloalkylalkyl, unsubstituted or substitutedheterocyclyl, unsubstituted or substituted heterocyclylalkyl,unsubstituted or substituted aralkyl, unsubstituted or substitutedheteroaryl, or unsubstituted or substituted heteroaralkyl; R₂ isunsubstituted or substituted alkyl, unsubstituted or substituted aryl,or unsubstituted or substituted heteroaryl; and R₃ is unsubstituted orsubstituted alkyl, unsubstituted or substituted aryl, unsubstituted orsubstituted cycloalkyl, unsubstituted or substituted cycloalkylalkyl,unsubstituted or substituted heterocyclyl, unsubstituted or substitutedheterocyclylalkyl, unsubstituted or substituted aralkyl, unsubstitutedor substituted heteroaryl, or unsubstituted or substitutedheteroaralkyl; with the proviso that at least one of R₁ and R₃ is otherthan unsubstituted aralkyl, or R₂ is other than unsubstituted aryl. 2.The compound of claim 1, wherein; R₁ is a substituted or unsubstitutedgroup selected from the group consisting of phenyl, naphthyl cyclohexyl,cycloheptyl, cyclooctyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl,bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.2.2]nonyl,bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decanyl, adamantyl, noradamantyl,bornyl, or norbornyl; pyrrolidinyl, imidazolidinyl, pyrazolidinyl,pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl,piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl,quinuclidyl, indolyl, indolinyl, adeninyl, guaninyl, quinolinyl,isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, —(C₁-C₈alkyl)phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, andbenzothiophenyl; R₂ is a substituted or unsubstituted group selectedfrom the group consisting of C₁-C₈ alkyl, phenyl, naphthyl, and pyridyl;and R₃ is a substituted or unsubstituted group selected from the groupconsisting of C₁-C₈ alkyl, —(C₁-C₈ alkyl)phenyl, and —(C₁-C₈alkyl)pyridyl.
 3. The compound of claim 1, wherein R₁ is a substitutedor unsubstituted group selected from the group consisting of phenyl,bicyclo[2.2.1]heptyl, benzyl, and morpholinyl.
 4. The compound of claim1, wherein R₂ is a substituted or unsubstituted group selected from thegroup consisting of propyl, butyl, and phenyl.
 5. The compound of claim1, wherein R₃ is a substituted or unsubstituted group selected from thegroup consisting of propyl, butyl, and benzyl.
 6. The compound of claim1, wherein the compound of Formula I is selected from the groupconsisting of:


7. The compound of claim 1, wherein the compound of Formula I isrepresented by Formula III:

wherein R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉,R₂₀, and R₂₁ are individually selected from the group consisting of H,F, Cl, Br, SO₂, NO₂, OH, NH₂, and substituted or unsubstituted C₁-C₈alkyl, with the proviso that at least one of R₇, R₈, R₉, R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, and R₂₁ is other than H.
 8. Apharmaceutical composition comprising the compound of claim 1, astereoisomer thereof the compound of Formula I, a pharmaceuticallyacceptable salt of the stereoisomer of the compound of Formula I, apharmaceutically acceptable salt of the compound of Formula I, acompound of Formula II, a pharmaceutically acceptable salt of thecompound of Formula II, or a mixture of any two or more thereof, and apharmaceutically acceptable carrier;


9. A method comprising inhibiting a cannabinoid receptor comprisingadministering the pharmaceutical composition of claim 8 to a subject.10. The method of claim 9, wherein the cannabinoid receptor iscannabinoid receptor subtype-2.
 11. A method comprising administeringthe pharmaceutical composition of claim 8 to a subject in need of ananti-inflammatory agent.
 12. A method comprising using the compound ofclaim 1, a pharmaceutically acceptable salt of the compound of FormulaI, a stereoisomer of the compound of Formula I, a compound of FormulaII, or a mixture of any two or more thereof as an active fluorescencedye control for a fluorometric binding assay;


13. A pharmaceutical composition comprising the compound of Formula II,a pharmaceutically acceptable salt of the compound of Formula II, or amixture thereof, and a pharmaceutically acceptable carrier;


14. A method comprising inhibiting a cannabinoid receptor comprisingadministering the pharmaceutical composition of claim 13 to a subject.15. The method of claim 14, wherein the cannabinoid receptor iscannabinoid receptor subtype-2.
 16. A method comprising administeringthe pharmaceutical composition of claim 13 to a subject in need of ananti-inflammatory agent.
 17. An active fluorescence dye control for afluorometric binding assay, the active fluorescence dye controlcomprising a compound of Formula II, a pharmaceutically acceptable saltof the compound of Formula II, or a mixture thereof;